Available online at http://www.journalcra.com INTERNATIONAL JOURNAL OF CURRENT RESEARCH International Journal of Current Research Vol. 5, Issue, 02, pp. 124-128, February, 2013

ISSN: 0975-833X RESEARCH ARTICLE

In vitro Determination of Marine Hyrtios erectus Secondary Metabolite Effect against Human Breast and Larynx Cancer Cell Lines

1Ramachandran, M. 2Titus Immanuel, 1Manley Backyavathy, P. and 1*Balwin Nambikkairaj

1Department of Zoology, Voorhees College, Thiruvalluvar University, Vellore, Tamil Nadu, India 2Fisheries Division, Central Agricultural Research Institute, Port Blair, A and N Islands, India

ARTICLE INFO ABSTRACT

Article History: Marine are rich sources of novel secondary metabolite and they are potential drug molecule to antitumor

Received 19th November, 2012 and antiprolific drug development. Hyrtios erectus was extracted and purified through different solvent fractions Received in revised form methods. Pure compound was obtained as brownish amorphous powder. The positive test for dragendorff reagent 24th December, 2012 was indicated as alkaloid group. Cytotoxicity was tested on Normal (Vero) cell line non tumor cells, Human Accepted 17th January, 2013 Breast cancer cell line (MCF-7) and Human epithelial larynx cancer cell line (Hep-2) using microculture Published online 14th February, 2013 tetrazolium (MTT) assay for anticancer activity. Sponge pure compound was non‐toxic to (Vero) cells but highly cytotoxicity to (53%) MCF-7 cells and low toxic to Hep-2(32%) was recorded at 25μg/ml concentration. Each concentrations express individual results of cells viability with cytotoxicity increase and decrease of their Key words: concentration level of pure compound. In this research further investigation require for this sponge purified Hyrtios erectus, compound in order to chemical structure elucidations as well as pre clinical study for anticancer activity.

Antitumor,

Cytotoxicity, Methanol extract, Zoochemical screening. Copy Right, IJCR, 2013, Academic Journals. All rights reserved.

INTRODUCTION different Hyrtios sp. and their associated microorganisms revealed the presence of numerous structurally unique natural products Marine sponge are rich source of structurally novel and biologically including scalarane sesterterpenes, acyclic triterpenes, indole active secondary metabolites considered to be true “chemical alkaloids and macrolides in addition to steroids. Many of these factories” producing hundreds of unique chemical compounds with compounds possess interesting biological activities. spongistatins, the different biological activities have been isolated. Sponges are simple, most important metabolites of the genus Hyrtios discovered to date, multicellular, sessile invertebrates with no true tissue layers or organ; showed powerful anticancer activity (Youssef, 2002; Pettit, 1998). they are cytotoxic, inhibit cell proliferation and could be used as The chemical investigations, which afforded the antineoplastic agents chemotherapeutics (Bergquist, 1978). These compounds differ sesterstatins (Longeon et al., 2011; Pettit et al., 1998). Cytotoxicity structurally and act on different cytoskeleton elements, but have assays are a widely used method in vitro toxicology studies. It is not similar anti prolific and antitumor activities. Moreover, many only rapid and standardized, but also a sensitive and inexpensive antitumor drug isolated and their structure determined, but their method to measure drug induced alterations in metabolic pathways or biological roles and activities are still largely unknown (Bacus and structural integrity which may or may not be related to directly to cell Green, 1974; Kornprobst,2005). Hyrtios erectus synonym H.erecta death (Pettit and Tan, 2005; Habib and Karim, 2011). Recent (: Porifera, class Demospongiae, order developments in the field of marine antitumor research with emphasis Dictyoceratida, family Thorectidae) is an colonies, sedentary, on the signal transduction pathways of oncogenesis, clinical status of grayish in color, irregularly rounded, asymmetrical, attached to sea the marine derived antitumor compounds and discuss the potential bottom by means of masses of spicules. challenges and probable strategies to combat the gap between marine compound isolation and successful antitumor drug development. These colonies are attached on the dead coral stones in shallow water inter-tidal areas at the depth of 10-20m. Sponge secondary metabolites show interesting biological activities, for example MATERIALS AND METHODS calyculins from calyx (Sipkema et al., 2005), discodermolide from D. dissolute (Kato et al., 1986), latrunculins Sponge collection from Latrunculia magnifica (Gunasekera, 1990; Kashman, 1980; Kashman et al., 1985; Spector, 1983), and spongistatins from Sponge tissue was collected by scuba divers at (10-20m) deep along Spongia sp. and Spirastrella sp. (Spector, 1989; Petit, 1993). The the South Andaman Sea coast (North Bay and Pongibalu secondary metabolites from the genus Hyrtios sp particularly region11°30’N and 92°39’E). The sponge collection was made in the Hertios erectus synonym H.erecta have been investigated extensively month of June, 2011. Samples were kept instead plastic packs in ice (Petit et al., 1994; Kobayashi et al., 1994; Miyaoka, 2000; Pettit boxes before freezing at -20°C until analysis. Sample was preserved et al., 1998; Yonghong, 2007). Previous chemical investigations of in 100% methanol solvent. Taxonomy details were identified and deposited at the Central agricultural research institute, Fisheries *Corresponding author: [email protected] division, Port Blair. A voucher specimen is under the accession number (PB5985).

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Extraction/Fractionation/Isolation method of plots of the percent of cell viability against the concentration of the tested compounds. The fresh sponge Hyrtios erectus (3kg weight) was chopped into small pieces and filled in glass containers soaked in methanol RESULTS (3 x 2.5L) which was shacked well twice daily around one weak. Methanol extract was filtered by No.1 Whatman filter paper (GE The pure compound was isolated from marine sponge ‘hexane’ with Healthcare, 125mm, UK) pooled into rotary evaporator (STRIKE ‘chloroform’ combined fraction and brownish amorphous powder 202, Germany). Methanol extract was concentrated under reduced o respectively. The positive tests of the pure compound are determined pressure 45 C, which was further dried under high vacuum (42g). by the method of dragendorff reagent which was identified alkaloid The residual mass was further extracted with methanol-chloroform group. In this research three cancer cell lines are (Vero) normal cell (80:20%). Complete drying the methanol-chloroform extract, a line non tumor cells, (MCF-7) Human Breast cancer cell line and residual mass of (23g) was obtained. Methanol extract showing (Hep-2) Human epithelial larynx cancer cell line selected for promising activity in vitro models, it was fractionated into four anticancer assays. In this study, the normal Vero cell line expressed fractions, i.e. the hexane(2g), chloroform(4g), n-butanol (6g)soluble 91% cells viability and 8% of inhibition of cancer cells was found at and n-butanol (11g) insoluble fractions. The hexane fraction was low concentration 25mg/ml of pure compound are shown in Table.1. found to be a complex mixture and was of low yield. Moreover, The (MCF-7) breast cancer cell line low viability 46% and cytotoxic hexane and chloroform fractions were mixed combined fraction was 53% was observed, even though 14% of viability and 85% of included for purification of its components was achieved by cytotoxic in 500mg/ml concentration level was recorded (Fig.1-2). chromatography over column of silica gel. One pure compound was Similarly result of (Hep-2) cancer cell line cells viability 67% and obtained after re-chromatography over flash silica gel (230-400 cytotoxicity 32% was obtained at 25mg/ml concentration. Low mesh) of the combined chloroform and hexane fraction (Lakshmi et percentage of viability 20% and high cytotoxicity 70% was found at al., 2012). Thin Layer Chromatography (TLC) analysis was carried 500mg/ml concentration on (Hep-2) cell line. The pure compound out for identifying purified compound spot using aluminium sheet was expressed potent cytotoxic activity 53% at 25mg/ml against precoated silica gel 60 F254 or on glass precoated RP-18 F254 plates (MCF-7) cancer cell line than Hep-2 cancer cell line. Minimum (Merck, Darmstadt, Germany). effective concentration of pure compound of sponge was non‐toxic to (Vero) cells but highly toxic to 53% (MCF-7) cells and low toxic Zoo chemical analysis to 32% (Hep-2) cells were recorded(IC50) at 25μg/ml concentration of the pure compound (Fig. 3-4). Pure compound was highly potent The Zoochemical analysis absence of an established protocol for the effect on cervical cancer cells could be inhibit on the cells screening of chemical constituents in animal tissues, the qualitative proliferative activity. Similarly increase the dose could increase the analysis for the possible bioactive components present in the sponge activity on cancer cells is dose dependent manner. In the presence of samples were done using established phytochemical screening 400 mg concentration of compound titrated on cancer cells protocol (Edeoga , 2005). contribute low percentage cells viability and high

Cytotoxicity Evaluation

The cell lines Human Breast cancer(MCF-7), Human epithelial larynx cancer(Hep-2) and Non tumourous normal cells(Vero) were obtained from American type culture collection (ATCC) Manassas, VA, and cultured in Dulbecco’s modified eagle’s medium supplemented with10% fetal bovine serum (FBS), 2mM glutamine and 100 mg/ml Penicillin-Streptomycin incubated at 37oC, humidified atmosphere with 5% CO2. Sponge purified compound was subjected into invitro analysis of cytotoxicity using MTT {(3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (Sigma, St. Louis, USA)} assay. Obtained cells were grown at concentration of 5 x 103 cells/well in 96 well plates. After24 h, cells were washed with fresh medium and were treated with different concentrations of crude extract (25, 50, 100 and 500mg/ml). The cells without the addition of sponge purified compound were taken as control. After 24 h incubation, 100 ml of MTT solution was added and further incubated for4 h at 37 oC. Eventually 100 ml DMSO was Fig. 1. Cells viability interpretation of Vero and MCF-7 cell line added to solubilize the formazan salt formed and the amount of formazan salt was determined by measuring the OD at 540 nm using microplate reader. The relative cell viability was determined by the amount of MTT converted into formazan salt. Viability of cells was quantified as a percentage compared to that of control. The experiment was carried out in triplicate and the data were expressed as mean from these three sets of experiments (Chairman, 2012).

% Viability = (Test OD Value / Control OD value) X 100

Statistical Analysis

The experimental data were expressed as mean ± sd. The significance of difference among the various treated groups and control group were analyzed by means of one‐way ANOVA. The level of significance was set at p < 0.05. IC50 (inhibitory concentration which

caused 50% inhibition) were estimated using linear regression Fig. 2. Cells viability interpretation of Vero and Hep-2 cell line

126 International Journal of Current Research, Vol. 5, Issue, 02, pp. 124-128, February, 2013

Table 1. Cells viability assay with sponge pure compound against normal (Vero) cell line, Breast Cancer cell line (MCF -7) and Human Epithelial

Larynx cancer cell line (Hep-2)

Non tumorous -Normal cell line (Vero) Human Breast Cancer cell line (MCF-7 ) Human Larynx Cancer cell line

Sponge Pure (Hep-2)

compound Optical Density Cells Cytotoxicity Optical Cells Cytotoxicity Optical Cells Cytotoxic Concentration value (OD) viability (%) Density viability (%) Density viability ity (%) mg/ml (%) value (OD) (%) value (OD) (%) Control 0.956±0.03 100 0 1.532±0.12 100 0 0.899±0.06 100 0

25 0.876±0.03 91.63 8.36 0.720±0.04 46.99 53.00 0.605±0.06 67.29 32.70 50 0.769±0.04 80.43 19.56 0.655±0.05 42.75 57.24 0.557±0.02 61.95 38.04 100 0.731±0.06 76.46 23.53 0.512±0.06 33.42 66.57 0.501±0.03 55.72 44.27 200 0.697±0.05 72.90 27.09 0.388±0.05 25.32 74.67 0.423±0.04 47.05 52.94 300 0.652±0.03 68.20 31.79 0.326±0.04 21.27 78.72 0.355±0.03 39.48 60.51 400 0.605± 0.04 63.28 36.71 0.245±0.02 15.99 84.00 0.275 ±0.03 30.58 69.41 500 0.513±0.05 53.66 46.33 0.218±0.01 14.22 85.77 0.187±0.04 20.80 79.19

Fig. 3. Cells Inhibition analysis of Vero and MCF-7 cell line

B

Fig.4. Cells Inhibition analysis of Vero and Hep-2 cell line

C

A

D 127 International Journal of Current Research, Vol. 5, Issue, 02, pp. 124-128, February, 2013

compound. Low dose of pure compound contributed less toxic against cancer cells as well as increase the dose of drug could be observe the high toxic of the cancer cells. Breast and larynx cancer cells could be prevent anti proliferative effects by the potent anti cancer drug of marine sponge pure compound. Previously literatures proved Sponge Hyrtios erecta secondary metabolite alkaloid compound prevent the human (HeLa) cervival cancer cell line, HCT- 116 colon carcinoma cell line, lymphoma cancer cell line (Mohamed et al., 2007). A biochemical study revealed that this compound inhibited myeloid leukemia cell differentiation protein Mcl-1, a critical player involved in survival and death decisions of individual cells (Michels and Packham, 2005). The alkaloid compound which has been isolated from various marine sponges revealed cytotoxic effect against AsPC-1 pancreatic cancer cells (Guzman et al., 2010). In the present study, we found that extracts from Hyrtios erecta exhibited significant anti-proliferative effects against a variety of human cancer cell lines. Further investigation require as well as

E essential to this sponge purified compound in order to chemical structure elucidations, characterization as well as pre clinical study for anticancer activity. In conclusion, it is believed that a rich source of anticancer drug candidates could be obtained from marine sponge secondary metabolites. To our knowledge, the results describe antitumor activity of marine sponge secondary metabolites promising to convey antitumor drug development. There was no statistical significant difference between control viability and treated cells.

Aknowledgement

The Authors are thankful to Director, Central Agricultural Research Institute, Port Blair, for providing necessary research facilities. We are greateful to Principal, Voorhees College, Vellore, Tamilnadu, for support to this research work.

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